This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall hypothesis of this proposal is that arthropod-expectorated proteins modulate the establishment and course of arboviral infections in the vertebrate host. Our working hypothesis is that dissemination of dengue virus to the salivary glands of Aedes aegypti mosquitoes modifies protein expression in the salivary glands and the expectorated saliva, thereby enhancing the transmissibility of the virus to people. This hypothesis is based on the requirement that dengue virus must disseminate into the salivary glands in order to be transmitted by the primary mosquito vector, Ae. aegypti [1] and that mosquito saliva can alter the local environment at the bite site in a way that encourages the establishment of an infection [2]. We infer then that some salivary components, injected into the vertebrate upon feeding, modulate the dengue infection in the human host. Our preliminary findings suggest that dengue infection alters the protein profile of the mosquito salivary glands. The sequencing and identification of these proteins and their possible roles in transmission remains to be investigated. Dengue virus has been shown to bind Ae. aegypti salivary gland and midgut proteins [3-4] and that there is at least one dengue virus receptor in these tissues [5]. However, whether there are virus binding proteins in the expectorated saliva, which could assist in the chaperoning of virus to target cells for the establishment of infection, has not been investigated. The saliva of mosquitoes contains a diverse cocktail of pharmacologically active compounds that are deposited simultaneously with virus to the bite site of the vertebrate host [6]. Some of these are responsible for the disruption of the homeostasis of human lymphocytes [7-8] and dendritic cells [9] in addition to eliciting saliva-specific antibody production [10]. How such cytokine modulation and preexisting antibodies to salivary proteins affect transmission of dengue from the vector to the vertebrate remains to be studied. We propose to investigate the specific changes that dengue infection renders on the salivary gland protein profile of the mosquito vector and how these changes affect the establishment of infection in vertebrate cells. Furthermore we will evaluate the immune response of the vertebrate to Ae. aegypti salivary proteins and whether this response alters the course of dengue virus infection. Specific Aims Aim 1: To characterize differential protein expression of saliva and salivary gland extract from Ae. aegypti between dengue-infected and uninfected mosquitoes. H0: Dissemination of dengue virus to the mosquito salivary glands changes the protein profile of the saliva and salivary glands. Aim 2: To identify proteins in mosquito saliva and salivary gland extract capable of binding to dengue virus and human immune cells such as lymphocytes and dendritic cells (DC). H0: Mosquito salivary proteins differentially expressed based on infection status may bind dengue virus and human lymphocytes, thereby enhancing the potential for dengue to establish replication in target immune cells. Aim 3: To characterize the vertebrate host immune response to Ae. aegypti salivary proteins, and the impact of that response on dengue infection. H0: Innate immune responses and preexisting antibodies to mosquito salivary proteins affect the transmission success of dengue from the mosquito vector to the vertebrate. Dengue virus infection in the mosquito alters these immune responses through down regulation of salivary proteins.